sign in sign up
<<
Home , Leptospirosis

DISPATCHES

Value of PCR, Serology, and Blood Smears for Human Granulocytic Anaplasmosis Diagnosis, France

Yves Hansmann, Benoit Jaulhac, Pierre Kieffer, a low sensitivity (3); PCR and serology are more widely Martin Martinot, Elisabeth Wurtz, Régis Dukic, available, but their diagnostic value is not well established. Geneviève Boess, André Michel, The aim of our study was to compare the diagnostic values Christophe Strady, Jean François Sagez, of the available microbiological tests in a prospectively se- Nicolas Lefebvre, Emilie Talagrand-Reboul, lected series of patients with clinical Xavier Argemi, Sylvie De Martino consistent with an HGA diagnosis.

We prospectively examined the effectiveness of diagnos- The Study tic tests for anaplasmosis using patients with suspected In this prospective, multicenter study, we enrolled symp- diagnoses in France. PCR (sensitivity 0.74, specificity 1) tomatic patients living in Alsace, a region of northeastern was the best-suited test. Serology had a lower specificity but higher sensitivity when testing acute and convalescent France where tickborne diseases are highly . Pa- samples. PCR and serology should be used in combination tients gave written, informed consent to participate in our for anaplasmosis diagnosis. study, which was approved by the ethics committee of the University Hospital of Strasbourg (Strasbourg, France). We included patients if they had 1 of the following uman granulocytic anaplasmosis (HGA) is a tickborne combinations of signs and symptoms occurring no more Hintracellular bacterial infection caused by Anaplasma than 4 weeks after a tick bite: 1) or other symptom phagocytophilum. The disease is present in North America, presumed related to a tick bite, 2) fever plus thrombocyto- Europe, and northern Asia, areas with Ixodes ricinus ticks, penia with or without or elevated liver enzyme the primary vector for transmission to humans (1,2). Clini- levels, 3) with or without leukopenia, cal manifestations of disease include acute fever, , or 4) elevated liver enzyme levels without fever. The first and occurring 2–3 weeks after tick bite. Diagnosis visit included clinical and epidemiologic evaluations and requires the isolation of A. phagocytophilum in blood cul- the collection of blood samples for A. phagocytophilum ture, the presence of morulae in polymorphonuclear cells serology, May Grünwald-Giemsa staining, and A. phago- after May Grünwald-Giemsa staining of peripheral blood cytophilum–specific PCR. We did not culture forA. phago- smears, positive serologic results (seroconversion or high cytophilum. An etiologic investigation was also conducted titer of specific ), or a positive A. phagocytophi- to obtain a . After >4 weeks, a sec- lum PCR result. The May Grünwald-Giemsa stain test has ond visit was scheduled to obtain a clinical evaluation, A. phagocytophilum serology, and (if necessary) a complete Author affiliations: Université de Strasbourg, Strasbourg, France differential diagnosis. (Y. Hansmann, B. Jaulhac, E. Talagrand-Reboul, S. De Martino); We stratified patients into 3 groups on the basis of Hôpitaux Universitaires de Strasbourg, Strasbourg their diagnosis. One group included controls, who were pa- (Y. Hansmann, N. Lefebvre, X. Argemi); Centre National de tients with a clinical and microbiologically confirmed non- Référence des , Strasbourg (B. Jaulhac, S. De Martino); anaplasmosis diagnosis. The second group included ana- Centre Hospitalier Emile Muller, Mulhouse, France (P. Kieffer); plasmosis patients defined by >1 if the following criteria: Centre Hospitalier Pasteur, Colmar, France (M. Martinot); intraleukocyte morulae on blood smears, a positive PCR Centre Hospitalier Saverne, Saverne, France (E. Wurtz); Centre result for Anaplasma, a 4-fold increased titer for Hospitalier de Haguenau, Haguenau, France (R. Dukic); Centre A. phagocytophilum in the follow-up sample or a serocon- Hospitalier de Guebwiller, Guebwiller, France (G. Boess); Centre version (i.e., change in antibody titer from negative in first Hospitalier de Wissembourg, Wissembourg, France (A. Michel); sample to >1:64 in second sample), or a high antibody titer Polyclinique Saint André, Reims, France (C. Strady); Centre for Anaplasma (>1:256) by indirect Hospitalier Sélestat, Sélestat, France (J.F. Sagez) antibody assay. The third group were patients without DOI: https://doi.org/10.3201/eid2505.171751 any diagnosis.

996 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 25, No. 5, May 2019 PCR and Human Granulocytic Anaplasmosis Diagnosis

We performed DNA extraction, PCR, and serologic specificity of 1 and blood smear staining had a sensitivity testing blinded to sample identification as previously de- of 0.21 and a specificity of 1. Seroconversion or a 4-fold scribed (4). The PCR targeted the A. phagocytophilum increase of antibody titer had a sensitivity of 0.32 and msp2/p44 gene. We performed serologic testing using specificity of 0.97, an antibody titer> 1:256 had a sensitiv- the Anaplasma phagocytophilum IFA IgG assay (Focus ity of 0.58 and specificity of 0.97, and overall serology Diagnostics, http://www.focusdx.com) (4). Trained staff had a sensitivity of 0.84 and specificity of 0.94. examined May Grünwald-Giemsa–stained smear prepa- The interval between the first and second serologic rations of whole blood samples for intracellular morulae. tests for most patients in the anaplasmosis group was 4–8 We collected data by using EpiData version 3.1.2701.2008 weeks (mean 49.8 days). Five patients had the second test (http://epidata.dk) and extracted data to Excel spreadsheets >8 weeks after the first. Of these patients, 2 seroconverted, (Microsoft, https://www.microsoft.com) for analysis. Af- 1 experienced a substantial decrease in antibody titer, 1 ex- ter patient stratification, we estimated the sensitivity and perienced a substantial increase at week 12, and 1 had a specificity of the different diagnostic tests. stable antibody titer. During May 2010–July 2012, we enrolled 155 patients Our study confirms PCR as the gold standard for diag- into the study, 25 of whom did not complete the second visit. nosis of HGA; this test enabled rapid diagnosis during the None of these 25 patients had a positive PCR result or an acute stage of infection with good sensitivity and excel- antibody titer >1:256 at the first visit. The remaining 130 lent specificity. However, the absence of a gold standard patients completed both study visits and were thus included diagnostic test to compare our results with is a limitation to in the study evaluation. Of these 130 patients, 19 had con- our study. A. phagocytophilum culture is the reference test firmed anaplasmosis diagnoses and 36 were controls with for HGA diagnosis (5,6) but is not well suited for routine confirmed nonanaplasmosis diagnoses (infections withBor - use because culturing is time-consuming and not widely relia burgdorferi, Epstein-Barr virus, , HIV, performed. The diagnosis of anaplasmosis often involves tick-borne encephalitis virus, spp., Babesia spp., assessing for the presence of morulae, but this test has low parvovirus B19, hantavirus, Francisella tularensis, Plasmo- sensitivity (3). In our study, this test was of limited value dium spp., and Aeromonas spp.). Of the patients with HGA, for HGA diagnosis because whenever morulae were de- 84.2% (16/19) met the serologic criteria and 73.7% (14/19) tected on blood smears >1 of the other diagnostic tests was met the PCR criteria (Table; Figure). Fever, the most fre- positive. However, May Grünwald-Giemsa staining is the quent symptom (89%), was associated with joint and muscle quickest test to do, and when performed by trained staff, pain. Cytopenia of platelets, neutrophils, or both (74%) and positive results are helpful for physicians. elevated liver enzyme levels (63%) were frequently present. In clinical practice, diagnosis of HGA often relies on Calculations of the diagnostic value of each test serology (7–9), but 2 limitations are associated with this method showed that PCR had a sensitivity of 0.74 and a method: a risk for false-negative results during the acute stage of infection because A. phagocytophilum antibodies

are detected on average 11.5 days after symptom onset and Table. Anaplasma phagocytophilum diagnostic test results of patients with nonanaplasmosis and human granulocytic a risk for false-positive results because Anaplasma antibod- anaplasmosis diagnoses, France, May 2010–July 2012 ies are detectable in 86.4% of patients for 6–10 months and Control group, Anaplasma in 40% of patients up to 2 years after the initial infection Test result no./total group, no./total Positive blood smear 0/36 4/19 (10). Positive serologic criteria are seroconversion, a 4-fold Positive by serology 2/36 16/19 increase in antibody titer, or a stable and high antibody titer Seroconversion* or 4-fold rise 1/36† 6/19‡§ in antibody titer Antibody titer >1:256 at first 1/36¶ 11/19§ visit Positive PCR 0/36 14/19 *Seroconversion is defined as a change in antibody titer from negative in the first sample obtained during acute illness to >1/64 in the second sample acquired >4 weeks later. †One patient had a seroconversion with a microbiologically confirmed diagnosis of parvovirus B19 infection. ‡Only 1 patient had a 4-fold increase in antibody titer, but the titer at the first study visit was already high enough to establish the diagnosis (increase from 1:512 to 1:2,048). §One patient had a seroconversion with an A. phagocytophilum antibody titer >1:256 at the second visit (patient counted once in both serology categories). All other patients with seroconversion had an antibody titer <1:256. Distribution of positive diagnostic test results for patients ¶One patient with microbiologically confirmed leptospirosis had an A. Figure. phagocytophilum antibody titer of 1:256 at the first visit that decreased to with confirmed human granulocytic anaplasmosis, France, May 1:64 at the second visit. 2010–July 2012.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 25, No. 5, May 2019 997 DISPATCHES

(11,12). In our study, we observed that each of these crite- Microbiol Infect Dis. 2012;72:214–8. http://dx.doi.org/10.1016/ ria can lead to misdiagnosis at the beginning of infection, j.diagmicrobio.2011.12.005 5. Goodman JL, Nelson C, Vitale B, Madigan JE, Dumler JS, as previously reported (13). Kurtti TJ, et al. Direct cultivation of the causative agent of human PCR is considered the most effective diagnostic granulocytic ehrlichiosis. N Engl J Med. 1996;334:209–15. method during early stage A. phagocytophilum infection http://dx.doi.org/10.1056/NEJM199601253340401 (14,15). Our results confirm this belief, despite our limita- 6. Wormser GP, Aguero-Rosenfeld ME, Cox ME, Nowakowski J, Nadelman RB, Holmgren D, et al. Differences and similarities tion of a small study population. However, if PCR is use between culture-confirmed human granulocytic anaplasmosis alone, HGA might be underdiagnosed. and early . J Clin Microbiol. 2013;51:954–8. http://dx.doi.org/10.1128/JCM.02929-12 Conclusions 7. Yeh MT, Mather TN, Coughlin RT, Gingrich-Baker C, Sumner JW, Massung RF. Serologic and molecular detection of granulocytic The presentation of fever in a patient with a history of tick ehrlichiosis in Rhode Island. J Clin Microbiol. 1997;35:944–7. bite does not qualify for an anaplasmosis diagnosis; mi- 8. Kocianová E, Kost’anová Z, Stefanidesová K, Spitalská E, crobiological tests need to be performed. For anaplasmo- Boldiš V, Hucková D, et al. Serologic evidence of Anaplasma sis, PCR testing appears to be the most effective diagnostic phagocytophilum infections in patients with a history of tick bite in central Slovakia. Wien Klin Wochenschr. 2008;120:427–31. tool. However, the sensitivity of PCR is <100%, and com- http://dx.doi.org/10.1007/s00508-008-1000-y bining PCR with serologic testing at the first visit appears 9. Aguero-Rosenfeld ME, Kalantarpour F, Baluch M, Horowitz HW, to be the best strategy for early diagnosis of acute anaplas- McKenna DF, Raffalli JT, et al. Serology of culture-confirmed mosis. In cases of high suspicion for HGA in patients with- cases of human granulocytic ehrlichiosis. J Clin Microbiol. 2000; 38:635–8. out any diagnosis at the first visit, a second serologic test 10. Lotric-Furlan S, Avsic-Zupanc T, Petrovec M, Nicholson WL, >4 weeks later can be helpful. A multiplex approach could Sumner JW, Childs JE, et al. Clinical and serological follow-up also be used in such cases to look for differential diagnoses. of patients with human granulocytic ehrlichiosis in Slovenia. Clin Diagn Lab Immunol. 2001;8:899–903. This study was supported by the French Hospital Clinical 11. Bakken JS, Haller I, Riddell D, Walls JJ, Dumler JS. The Research Program (PHRC HUS 2007–3960). serological response of patients infected with the agent of human granulocytic ehrlichiosis. Clin Infect Dis. 2002;34:22–7. http://dx.doi.org/10.1086/323811 About the Author 12. Brouqui P, Bacellar F, Baranton G, Birtles RJ, Bjoërsdorff A, Dr. Hansmann is head of the Infectious Disease Department at Blanco JR, et al.; ESCMID Study Group on Coxiella, Anaplasma, and Bartonella; European Network for Surveillance of Strasbourg University Hospital, Strasbourg, France; a member Tick-Borne Diseases. Guidelines for the diagnosis of tick-borne of the Borreliosis group of the European Society of Clinical bacterial diseases in Europe. Clin Microbiol Infect. 2004;10:1108– Microbiology and Infectious Diseases; and involved in 32. http://dx.doi.org/10.1111/j.1469-0691.2004.01019.x designing the tickborne disease national diagnosis and health 13. Lotrič-Furlan S, Rojko T, Jelovšek M, Petrovec M, Avšič-Županc T, Lusa L, et al. Comparison of clinical and laboratory characteristics plan for France. His research interests are tickborne diseases of patients fulfilling criteria for proven and probable human and diagnosis. granulocytic anaplasmosis. Microbes Infect. 2015;17:829–33. http://dx.doi.org/10.1016/j.micinf.2015.09.017 14. Dumler JS, Madigan JE, Pusterla N, Bakken JS. Ehrlichioses in References humans: epidemiology, clinical presentation, diagnosis, and 1. Strle F. Human granulocytic ehrlichiosis in Europe. Int J Med treatment. Clin Infect Dis. 2007;45(Suppl 1):S45–51. Microbiol. 2004;293(Suppl 37):27–35. http://dx.doi.org/10.1086/518146 2. Dedkov VG, Simonova EG, Beshlebova OV, Safonova MV, 15. Weil AA, Baron EL, Brown CM, Drapkin MS. Clinical findings Stukolova OA, Verigina EV, et al. The burden of tick-borne and diagnosis in human granulocytic anaplasmosis: a case series diseases in the Altai region of Russia. Ticks Tick Borne Dis. from . Mayo Clin Proc. 2012;87:233–9. 2017;8:787–94. http://dx.doi.org/10.1016/j.ttbdis.2017.06.004 http://dx.doi.org/10.1016/j.mayocp.2011.09.008 3. Bakken JS, Dumler JS. Human granulocytic anaplasmosis. Infect Dis Clin North Am. 2015;29:341–55. http://dx.doi.org/10.1016/ Address for correspondence: Yves Hansmann, Hôpitaux Universitaires j.idc.2015.02.007 de Strasbourg, Médecine Interne et Maladies Infectieuses 4. Koebel C, Kern A, Edouard S, Hoang AT, Celestin N, Hansmann Y, et al. Human granulocytic anaplasmosis in eastern et Tropicales 1, Place de l’Hôpital, Strasbourg 67091, France; email: France: clinical presentation and laboratory diagnosis. Diagn [email protected]

998 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 25, No. 5, May 2019